SUMMARY OF WORK This research area involves the study of embryonic stem cells prior to and during differentiation to cardiomyocytes. For these studies, we employ embryonic stem (ES) cells (R1, D3), embryonic germ cells (EG-1) and embryonic carcinoma cells (P19). We have established one of the most efficient systems available for the generation of cardiomyocytes from ES cells in vitro, and we have established when the ryanodine receptor, SR CaATPase, phospholamban and dihydropyridine receptor were first expressed with in vitro differentiation, permitting studies of EC coupling with differentiation. Selection protocols (most recently with a cardiac-restricted portion of the Na/Ca exchanger promoter) have permitted the isolation of cardiomyocytes. Overall, the research is aimed at generating cardiac-lineage specific cells to understand the role of regulatory proteins in the formation of cardiomyocytes in vitro. Additionally, if the cells can be isolated to homogeneity and shown to be viable after in vitro differentation, then we propose using this technique to test delivery protocols and improvements of cardiac function following cellular based therapies in rodent. A major focus of this basic research effort is devoted to the improved viability of these cells during in vitro cultivation conditions. Several target genes are also being modified in the stem cells to determine their role in cardiomyocyte differentiation and survival, and more recently, we have begun isolating (and targeting) cells to select sub-populations of cardiac progenitor cells that may be more appropriate for cellular based therapies. By studying the basic biology of embryonic stem cells, we hope to delineate mechanisms responsible for cardiomyocyte development and renewal, and apply these results to improve cellular based therapies that may be applicable to man.